Cellular proteins are essential both for virus replication and cellular defense. A key mechanism for regulation is the post-translational addition of chemical groups (e.g., phosphorylation, acetylation) onto either host or viral proteins. This proposal focuses on the under-studied protein modification ADP-ribosylation and the ability of the alphavirus nonstructural protein (nsP) 3 macrodomain (MD) to functionally interact with and regulate ADP- ribose (ADPr)-modified proteins. ADP-ribosylation is the addition of ADPr units onto proteins by ADP- ribosyltransferases, including a 17-member family commonly known as poly(ADPr) polymerases (PARPs). This modification can be recognized and reversed by some MDs. The MD protein fold binds ADPr and is highly conserved from viruses to archaea to humans. Viral MDs are present within the nsPs of a subset of plus-strand RNA viruses - alphaviruses, rubella virus, coronaviruses and hepatitis E virus. Alphaviruses are mosquito- borne viruses with an expanding range that cause outbreaks of rash, arthritis and encephalomyelitis. Within a few hours after infection, alphaviruses inhibit host protein synthesis and induce formation of stress granules (SGs) that are disassembled by nsP3. Alphavirus nsP3 has a N-terminal MD that is a determinant of neurovirulence and we have shown that the nsP3 MD (nsP3MD) not only binds but also hydrolyzes (removes) ADPr groups from ADP-ribosylated substrates. Mutation of the nsP3MD showed that replication and virulence of chikungunya virus (CHIKV) depend on these interactions. CHIKV nsP3MD mutants without binding or hydrolase activity are not viable and rapidly revert the mutations to wild type. Mutants with reduced activity can be recovered, but replicate less well in neural cells and have decreased virulence in mice. However, the ADP- ribosylated substrates recognized by viral MDs and their role(s) in virus replication have not been identified. We have developed a non-biased proteomics approach to identify ADP-ribosylated sites and substrates and defined the specificity of the CHIKV MD hydrolase activity as ADP-ribosylated aspartate and glutamate, but not lysine, residues. Preliminary data suggest that both nsP3MD binding to ADP-ribosylated proteins and hydrolase activities are necessary for efficient replication including initiation of infection and disassembly of SGs - cytoplasmic structures that sequester translation initiation factors. We hypothesize that alphaviral MDs regulate virus replication and the function of ADP-ribosylated proteins by binding to and/or removing ADPr groups from specific proteins at different stages of the replication cycle. To test these hypotheses and identify the interacting proteins using our novel proteomics approaches, we will: (1) Determine the step(s) during initiation and amplification of alphavirus replication that requires ADPr binding and/or hydrolase activities of the nsP3MD and (2) Determine the role of nsP3MD ADPr binding and hydrolase activity in SG disassembly and in translation of the subgenomic viral RNA during late stages of virus replication.